Osteoporosis and cancer-associated bone disease cause vast morbidity and mortality and are major health threats. The magnitude of these problems has generated considerable interest in understanding the molecular mechanisms that regulate bone density and bone formation. Identification and characterization of the genes that regulate osteoblast proliferation and differentiation have provided new insights into how bone formation occurs. Runx2 is a transcription factor-encoding gene that is necessary for osteoblast development and differentiation. Runx2 transgenes and polymorphic alleles are linked to osteopenia and fracture risk. Runx2 activity is tightly controlled by many cellular, molecular and intermolecular mechanisms. In spite of this vast knowledge, there is still a critical gap concerning how Runx2 activity is negatively controlled. The long-range goal of this work is to understand the molecular mechanisms that control Runx2 activity in osteoblasts for therapeutic purposes. The objective of this application is to determine how the DNA-binding protein and Wnt effector LEF1 regulates Runx2. The central hypothesis is that LEF1 inhibits Runx2-dependent transactivation and osteoblast differentiation. This hypothesis will be tested by pursuing these three Specific Aims: (1) Determining that LEF1 is required for osteoblast maturation with RNA interference and by etopically expressing gain-of-function and loss-of-function LEF1 proteins, (2) Defining the mechanism(s) by which LEF1 represses Runx2 transcriptional activity, and (3) Determining how molecules upstream of LEF1 in the canonical Wnt signaling pathway affect Runx2 transcriptional activity. These results are expected to describe molecular mechanisms from which novel therapeutic interventions for abnormal bone mineral density will be derived. In addition, it is expected that the results will fundamentally advance the fields of transcriptional regulation and osteoblast biology.